10424 • The Journal of Neuroscience, December 5, 2018 • 38(49):10424–10437 Development/Plasticity/Repair Congenital Cytomegalovirus Infection Alters Olfaction Before Hearing Deterioration In Mice X Franc¸oise Lazarini,1,2 Lida Katsimpardi,1,2* Sarah Levivien,1,2,3*Se´bastien Wagner,1,2 XPierre Gressens,3,4,5 Natacha Teissier,3,4,6† and Pierre-Marie Lledo1,2† 1Institut Pasteur, Perception and Memory Unit, F-75015 Paris, France, 2Centre National de la Recherche Scientifique, Unite´ Mixte de Recherche 3571, F-75015 Paris, France, 3PROTECT, INSERM, Unite´ 1141, F-75019 Paris, France, 4Paris Diderot University, Sorbonne Paris Cite´, F-75018 Paris, France, 5Center for Developing Brain, King’s College, London, WC2R2LS United Kingdom, and 6Pediatric Otorhinolaryngology Department, Robert Debre´ Hospital, Assistance Publique–Hoˆpitaux de Paris, F-75019 Paris, France In developed countries, cytomegalovirus (CMV)-infected newborns are at high risk of developing sensorineural handicaps such as hearing loss, requiring extensive follow-up. However, early prognostic tools for auditory damage in children are not yet available. In the fetus, CMV infection leads to early olfactory bulb (OB) damage, suggesting that olfaction might represent a valuable prognosis for neurological outcome of this viral infection. Here, we demonstrate that in utero CMV inoculation causes fetal infection and growth retardation in mice of both sexes. It disrupts OB normal development, leading to disproportionate OB cell layers and rapid major olfactory deficits. Olfaction is impaired as early as day 6 after birth in both sexes, long before the emergence of auditory deficits. Olfactometry in males reveals a long-lasting alteration in olfactory perception and discrimination, particularly in binary mixtures of monomolecular odorants. Although sensory inputs to the OB remain unchanged, hallmarks of autophagy are increased in the OB of 3-postnatal week-old mice, leading to local neuroinflammation and loss of neurons expressing tyrosine hydroxylase and calbindin. At the cellular level, we found CMV-infected cells and an increased number of apoptotic cells scattered throughout the OB layers, whereas cell proliferation in the neurogenic subventricular zone was decreased. These cellular observations were long-lasting, persisting up to 16 weeks after birth in both males and females and thus providing a mechanism supporting olfactory loss. Despite obvious differences in neurogenesisbetweenhumanandmouse,thesefindingsoffernewstrategiesaimedatearlydetectionofneurologicaldysfunctionscaused by congenital infections. Key words: congenital; cytomegalovirus; deafness; hyposmia; interneuron; olfactory bulb Significance Statement In developed countries, congenital cytomegalovirus (CMV)-infected newborns are at high risk of developing sensory handicaps such as hearing loss, thus requiring prolonged follow-up. In this study, we describe for the first time the functional impact of congenital CMV infection on the olfactory system and its associated sense of smell. We demonstrate that a mouse model of congenital CMV infection shows defects in olfactory bulb (OB) normal development and pronounced olfactory deficits affecting acuity and discrimination of odorants. These major olfactory deficits occur long before the emergence of auditory deficits through the upregulation of OB autophagy inducing local neuroinflammation and altered neuron content. Our findings provide new opportunities for designing olfactory means to monitor the possible neurological outcome during congenital CMV infection. Introduction sible sequelae in the fetus and newborn and the absence of The high seroprevalence of herpes virus type-5 or cytomegalovi- vaccines or curative treatments (Teissier et al., 2011; Manicklal et rus (CMV) constitutes a major public health concern due to pos- antibody against murine CMV used in this study; Yoann Madec (Emerging Diseases Epidemiology Unit, Institut Received March 21, 2018; revised Sept. 24, 2018; accepted Oct. 10, 2018. Pasteur, Paris) for help in conducting the statistical analysis; the Imagopole-Plateforme d’Imagerie Dynamique Author contributions: F.L., L.K., N.T., and P.-M.L. wrote the first draft of the paper; F.L., N.T., and P.-M.L. edited (PFID) France–BioImaging infrastructure, supported by the French National Research Agency (ANR 10-INSB-04-01, the paper; F.L., N.T., and P.-M.L. designed research; F.L., L.K., S.L., and N.T. performed research; S.W. and P.G. Investments for the Future), for advice and access to the CV1000 system; and Zeina Serhal, Laurent Cotter, and contributedunpublishedreagents/analytictools;F.L.,L.K.,S.L.,andN.T.analyzeddata;F.L.,N.T.,andP.-M.L.wrote Be´atrice de Cougny for technical help during the course of this study. the paper. The odorant mixtures are the subject of a patent application (EP3245944 published on November 22, 2017) by This work was supported by the Institut Pasteur, Paris (GPF 2015 Microbes and Brain “INFECSMELL”), AG2R-La- Institut Pasteur, Centre National de la Recherche Scientifique, and Assistance Publique–Hoˆpitaux de Paris on which Mondiale, Inserm Paris VII, and Fondation de l’Avenir (ET4718). We thank Professor Stipan Jonjic´ for providing the F.L., P.-M.L., N.T., and S.L. are named as inventors. The remaining authors declare no competing financial interests. Lazarini et al. • Early Olfactory Deficits in Congenital CMV Infection J. Neurosci., December 5, 2018 • 38(49):10424–10437 • 10425 al., 2013). CMV is transmitted through bodily fluids, likely by the impairment might represent a valuable prognostic tool for oral and/or the intranasal route (Farrell et al., 2016; Jackson and these congenital infections. Sparer, 2018). Infection is usually asymptomatic or can have a To address the impact of CMV on olfaction, we investigated flu-type presentation except when transmitted from mother to olfactory behavior in an animal model of congenital CMV infec- fetus. CMV belongs to the TORCH class of agents affecting the tion (Sakao-Suzuki et al., 2014). We show that virus-infected fetal/neonatal brain that are transmitted in utero or intrapartum. mice display OB development abnormalities and pronounced These agents include toxoplasmosis, rubella, herpes simplex and, olfactory deficits long before the emergence of auditory deficits. recently, Zika virus (Coyne and Lazear, 2016). In developed These findings provide the rationale for designing new olfactory countries, CMV infection is the leading cause of congenital mal- tests to monitor the possible neurological outcomes of these viral formations. During pregnancy, CMV infects the placenta with infections. varied outcomes ranging from fetal growth retardation to irre- versible birth defects, depending on maternal immunity and ges- Materials and Methods tational age (Pereira and Maidji, 2008; Fornara et al., 2017). Very Ethics statement. All animal procedures were performed in accordance few prognostic tools for neurosensory sequelae have been devel- with French legislation and in compliance with the European Commu- oped; these include ultrasound examination for macroscopic nities Council Directives (2010/63/UE, French Law 2013–118, February brain abnormalities and viral burden at birth (Forner et al., 6, 2013) according to the regulations of INSERM and Pasteur Institute 2015). Approximately 0.5–2% of newborns are CMV infected Animal Care Committees. The Animal Experimentation Ethics Commit- and are at high risk of developing learning disabilities and hearing tee (CETEA 89) of the Pasteur Institute approved this study (2015– 0028). Mice were housed in isolators and manipulated in class II safety loss, requiring prolonged follow-up care (Williamson et al., 1992; cabinets in the Pasteur Institute animal facilities accredited by the French Townsend et al., 2013). Ministry of Agriculture for performing experiments on live rodents. CMV exhibits particular tropism for neural stem cells of the CMV transplacental inoculation. Pups of timed Oncins France 1 (OF1) olfactory system of fetuses (van Den Pol et al., 1999; Odeberg et pregnant mice from Charles Rivers Laboratory were intraplacentally in- al., 2006; Teissier et al., 2014), thus lesioning the OB, but no study oculated under anesthesia (isoflurane) with 2 ␮l of murine CMV (Smith has hitherto examined the impact of the virus on olfaction. Other strain, ATCC VR-1399, at 10(4.75)TCID50/0.2 ml) or PBS at embryonic studies have reported olfactory deficits caused by neurotoxic and day 13 (E13) as described previously (Sakao-Suzuki et al., 2014). viral lesions (Lazarini et al., 2012, 2014; Tian et al., 2016). Because Design and groups. Following in utero intraplacental inoculation, mice ϭ olfactory cues guide many behaviors, most importantly feeding (total n 81) were divided in two groups, control (CTL) and CMV- and social behaviors especially at early stages in life, it is plausible infected mice (CMV) for two separate experimental sets. Experimental set 1. Experimental set 1 studied the impact of congenital that CMV induces behavioral changes resulting from olfactory CMV infection on growth, olfaction, olfactory epithelium (OE), OB, and defects. subventricular zone (SVZ) of preweaning mice (see Figs. 1C, 2, 5). This Olfaction is the first sensory modality to develop during fetal first set comprises a total of 56 inoculated mice (CTL: n ϭ 21, including life in mammals, long before vision and audition (Sarnat and Yu, 15 males; CMV: n ϭ 35, including 25 males). Olfaction was assessed 6 2016). Odor processing begins in olfactory sensory neurons and